The research described in this competing continuation application is related to the major aim of the original proposal: the assessment of cyclic nucleotide metabolism in primary culture of Sertoli cells with emphasis on the functional role of cyclic nucleotides in mediating hormonal effects. Biochemical and immunocytochemical approaches will be used to define the effect of FSH or cAMP-dependent protein kinase and Ca2 ion-calmodulin activity by monitoring cAMP-dependent and Ca2 ion-dependent endogenous phosphorylation of proteins as well as the characterization of cAMP binding proteins in subcellular fractions (nuclear, mitochondrial, plasma membrane, microsomal and microsomal-ribosoml) by immunoprecipitation. Immunocytochemical procedures (ferritin bridge and protein A-colloidal gold) will be carried out at the electron microscope level to determine the synthetic and secretory pathway of androgen-binding protein (ABP) together with the time-course of ABP secretion traced by time-lapse cinematography combined with ABP radioimmunoassay of the tissue culture medium. This study will provide a link between FSH and cyclic nucleotide effects on Sertoli cells and the secretory process of ABP, a biological marker for Sertoli cell function. Finally, we will characterize by two-dimensional gel electrophoresis and immunoautoradiography the FSH and cyclic nucleotide-dependent protein synthesis and secretion of ABP and other secretory proteins using Sertoli cell cultures established from rats of several ages to understand the possible relationship between age-dependent, hormonally-activated gene expression and initiation and establishment of spermatogenesis in the mammalian testis. The overall objective of this proposal is the understanding of the cell biology of Sertoli cell in vitro and the mechanism of regulation of spermatogenesis.